Name: a fast air-dry dropping chromosome preparation method suitable for fish in plants
Uploaded: 2015-12-16T14:00:00.000+00:00
Duration: 9 min 8 s
Description: Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1.染色体制备<ol><li>种子萌发和根尖的固定<ol><li>发芽在黑暗条件下的皮氏培养皿2天在22-24℃，在湿润的滤纸两层10-20大麦种子。通过用刀片切断剧烈根系1-2厘米从种子的长度。 </li><li>通过将装有冷自来水入碎冰 - 水的500ml玻璃瓶制备冰冷的水中。充入冰冷的水，沉浸根尖为20小时，以增加中期细胞的频率。 </li><li>从水到根部到50毫升乙醇:乙酸（3:1）固定液固定它们在室温2天。商店的根在新制备的乙醇:乙酸（3:1）固定液在4°C，直到使用最多的一年。 </li></ol></li><li>洗涤和酶处理<ol><li>洗10-20根用30ml冰冷的自来水进行5分钟，两次用50ml的玻璃烧杯中。使用双目显微镜。通过一个传输根一成30毫升使用镊子和通过摇动在玻璃烧杯5分钟洗两次的0.01M柠檬酸盐缓冲液（0.01M柠檬酸+ 0.01柠檬酸钠，pH值4.8）。放置在滤纸上的根，完全除去液体并用刀片切断不希望的非分生组织。 </li><li>孵育达于1ml酶混合物20根尖，在37℃下进行约50分钟，以软化植物组织（表1）在表玻璃。酶混合物含有0.7％的纤维素酶R 10，0.7％纤维素酶，1％果胶酶和1％cytohelicase稀释在0.01M的柠檬酸盐缓冲液。在-20℃下储存酶混合物和重复使用高达五倍。 </li><li>通过移液除去酶并用5ml的0.01M柠檬酸盐缓冲液洗根尖在冰上两次更换残留酶。 </li></ol></li><li>根浸渍<ol><li>洗涤根尖用1ml 96％乙醇仔细在同一表玻璃两次。更换新鲜配制的固定液（75％乙酸:25％乙醇），乙醇。使用10-15每根尖微升固定液。 </li><li>转印根尖连同固定剂到2ml管和崩解根分生组织用解剖针或镊子。挖掘管20次以重新悬浮细胞，以获得一个细胞悬浮液。存储所述细胞悬浮液在-​​20℃到两个月。 </li></ol></li><li>细胞悬浮液的滴液<ol><li>放置2-3层水渍薄纸在热板上在50℃下。沉浸显微镜载玻片在冰冷的自来水在冰箱30分钟。而地方上滑动的湿纸巾之上。 </li><li>吸管7-10微升细胞悬浮液，并从20厘米的距离拖放到冷却滑动放置在热板上。吸管上相同的位置上的滑动细胞悬浮液10微升乙酸 - 乙醇混合物中，并保持在热板上进行额外2分钟的幻灯片。放置在热板上滑动而不湿纸巾，并让它干燥1分钟。 </li></ol></li><li>质量控制和存储媒体Ë幻灯片<ol><li>检查使用相位差显微镜来控制染色体扩散的质量幻灯片。通过在-20℃下浸在96％的乙醇中，在科普林缸使用滑板或者在同一天或存储。 </li></ol></li><li>鱼类面前，幻灯片预处理;所有步骤都在室温下<ol><li>在含有50ml 2×SSC的科普林缸地方载玻片（20×SSC含有3M NaCl的和300mM的柠檬酸三钠）5分钟。使用镊子，转印滑动到含有3-10分钟加入50ml 45％乙酸的科普林缸。 </li><li>转移幻灯片包含10分钟50毫升2X SSC的科普林缸。转移到载玻片含有50ml 4％甲醛的科普林缸（在2×SSC），并浸泡载玻片10分钟，以固定的染色体。 </li><li>通过漂洗载玻片3次，每次4分钟，在含50ml 2×SSC中科普林缸清除甲醛。脱水在科普林缸载玻片2分钟在一系列的70％，90％和100％的乙醇，分别和干片在垂直位置。 </li></ol></li></ol> 2. 荧光原位杂交（FISH） <ol><li>对于每一个幻灯片，准备20微升杂交液共使用10微升去离子甲酰胺，5微升4×杂交缓冲液（200微升缓冲液中含有80微升20X SSC，8微升的1M的Tris-HCl pH值8.0，1.6微升0.5M的EDTA，11.2微升10微克/微升的鲑精和99.2微升DNA酶的水），3微升探针和2μl的DNA酶的水。 </li><li>加入20微升的每张幻灯片和盖杂交液用24×32毫米遮滑逮捕盖玻片用橡皮泥。同时变性幻灯片探针在80℃下，在热板2分钟。 </li><li>转移到幻灯片湿室和孵化的幻灯片，在37°CO / N避光。冲洗的幻灯片在科普林罐2X SSC取出盖玻片。在含55-60℃2×SSC和一个科普林缸地方幻灯片孵育20分钟。 </li><li>放置载玻片到2X SSC在科普林缸在室温2分钟。脱水的幻灯片在科普林缸为串联的分别为70％，90％和100％的乙醇，2分钟。 </li><li>风干载玻片和染液用1微克/毫升4&#39;​​，6-二脒基-2- phenylindoline吲哚（DAPI）在抗褪色封固剂，避免强光。 </li></ol> 3.微观分析和存储<ol><li>分析用落射荧光显微镜载玻片。过滤器的选择取决于用于探针标记的荧光染料。如果有必要，在4℃黑暗条件下长达一年的时间在商店的幻灯片。 </li></ol>

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The authors have nothing to disclose.

染色体制备实验已进行了使用属于禾本科（禾本科）谷物幼根。所有分析的品种有14相对较长的有丝​​分裂中期染色体（11-15微米）的二倍体基因组集，属于大型基因组的物种（5.1-7.9 GBP）的。 发芽根长度不超过2厘米，以获得最大的分生组织。分裂细胞同步是由一个20小时长冰-水处理该改进的有丝分裂中期的扩散量10来实现的。 两个步骤是在制备高品质的染色体制剂重要:（I）的50％的相对湿度-55％和酶处理的（Ⅱ）的持续时间。第一点是通过将湿纸组织在热板上在玻片的附近来实现。相对湿度的测定使用湿度计。对于植物的制备的最佳湿度染色体类似于报道基洛夫等人的湿度 。6。上处于最佳相对湿度的染色体质量的积极效应发生由细胞质和细胞壁水解的肿胀。 酶治疗的持续时间是物种依赖性的（ 表1）。酶处理的时间也取决于在乙醇/乙酸根固定和根的大小的时间跨度。较长的根已存储在固定液（高达1年，在4°C）中，时间越长消化根部到适当的等级。充分消化的根材料难以浸渍并会增加制剂的总时间作为长期持续浸渍的结果。此外，中期染色体保持嵌入到细胞质中，可能妨碍FISH实验过程中随之而来的探头渗透。另一方面过度消化材料可以影响染色体本身的结构，并破坏靶DN一种用于FISH分析。 用于制备的改善的另外一个因素是使用固定剂的第二降（3:1，乙酸/乙醇）的。高浓度的乙酸在该混合物的刺激细胞质的消化，并促进染色体中的物种与大染色体扩散。细胞质还原也可以在幻灯片染色体的固定化之后进行。为了这个目的携带染色体差显微镜载玻片可以在45％的乙酸中，在室温孵育2-10分钟，这取决于细胞质水平。用相位差显微镜进行染色体差的质量检查，没有任何补充染色（例如，1％的乙酰-CARMIN）。通常超过25幻灯片含有高品质的染色体差可以从使用​​上述方法20根来获得。 采用5S rDNA序列，[铁通] 10对有丝分裂中期染色体的FISH实验的结果，和6K的b长单拷贝探针（FPct_40752）分别为不同的高品质的上述（ 图2）中描述的所有物种和。这种方法的显而易见的好处是作为成功的FISH分析了完善的前提条件良好的蔓延，没有损坏和大量的中期染色体。能够以存储在细胞悬浮液在-​​20℃至两个月以制备染色体涂在FISH实验当天。新鲜制备的载玻片也可以储存在-20℃下在96％的乙醇，尽管我们观察到杂交信号上这种染色体的质量相比，新制备中期扩展减小。 制备由快速风干滴技术染色体差均适宜FISH和被再现的次数。该染色体的制备方法与FISH的组合可以被广泛地应用到探索在植物基因组中的组织，例如，对于核型11，彩色石印OMAL映射12，在合成的研究，以及对物理和遗传图谱13的集成。

荧光原位杂交（FISH）是一种有效的工具，单一的，高拷贝序列在染色体水平的物理图谱。前提条件是高品质的染色体利差的准备。没有一般染色体制备方案，这将是同样适用于动物和植物细胞。植物染色体的制备是特别具有挑战性，因为不同的物种中的刚性细胞壁和各种细胞质的一致性。之一的有利方法植物染色体的制备是一个所谓的滴技术也被称为落扩频技术和空气干燥技术1,2。该方法首先在1958引入由Rothfels和Siminovitch用于体外生长哺乳动物细胞3。后来Martin等人4和Kato等人5适于这种方法的植物。 最近，一个名叫法"SteamDrop＆＃39;被开发了用于水蒸汽的非重叠的染色体6的制备。虽然，前面7观察到高湿度的积极影响，&#39;SteamDrop&#39;提供高质量染色体制剂6的受控流。蒸汽处理引起拉伸可能连接到染色体蛋白质的一些修改染色体。导致中期扩展的质量是非常高的，虽然中期完成足够数量的固定利差随后FISH实验要求的专业技术。 在这里，我们提出了一个协议为适合FISH检测单和高仿探测器5,8的有丝分裂染色体谷物的准备。这种方法是由加藤下的50％-55％的相对湿度进行9（图1）中描述的风干落下法的改进的变体。这个协议包括减少数量的洗涤步骤使得其应用简便，高效和可重复性。使用这个协议，我们得到高品质的染色体利差和FISH结果大麦，H球茎，H海鱼，H murinum，H pubiflorum和黑麦 。

<table><tbody><tr><td>Hot Plate</td><td>MEDAX GmbH</td><td>12603</td><td></td></tr><tr><td>Cellulase R10</td><td>Duchefa</td><td>C8001</td><td></td></tr><tr><td>Cellulase&amp;nbsp;</td><td>CalBioChem</td><td>219466</td><td></td></tr><tr><td>Pectolyase</td><td>Sigma</td><td>P3026</td><td></td></tr><tr><td>Cytohelicase</td><td>Sigma</td><td>C8274</td><td></td></tr><tr><td>Texas Red-12-dUTP</td><td>Invitrogen</td><td>C3176</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluorecsence microscope</td><td>Olympus BX61</td><td>BX61</td><td></td></tr><tr><td>CCD camera</td><td>Orca ER, Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1200</td><td>fluorecsent dye</td></tr></tbody></table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

染色体利差的准备是荧光原位杂交技术的成功表现（FISH）的先决条件。高品质的植物染色体利差准备是具有挑战性，因为刚性细胞壁。之一的植物染色体的制备的批准的方法是所谓的滴制剂，也称为滴扩展或空气干燥技术。在这里，我们提出了一个协议，为快速编制有丝分裂染色体的利差适合FISH检测单和高仿DNA探针。这种方法是在50％-55％的相对湿度下进行的风干滴法的改进的变体。这个协议包括清洗步骤使得其应用简单，有效和可再现的数目减少。这种方法的显而易见的好处是作为成功的FISH分析了完善的前提条件良好的蔓延，没有损坏和大量的中期染色体。使用这个协议，我们得到高品质的CHROMosome差价和可重复的FISH结果大麦，H球茎，H海鱼，H murinum，H pubiflorum和黑麦 。

显微镜载玻片与有丝分裂的中期扩展是由上述的快速风干滴染色体制备方法制备（增刊，图1）。 FISH进行了分析，同时使用，重复和单拷贝序列。图像由一个落射荧光显微镜获得具有一组过滤器能够对应荧光团的激发和通过高灵敏度的CCD单色照相机捕获。用于图像采集，我们使用的计算机具有图像采集软件。采用5S rDNA序列，[铁通] 10和单拷贝探针的有丝分裂中期染色体上的荧光原位杂交实验的结果是独特的，高品质的大麦 （Figre 2A，B），H 球茎 （ 图2C），H。 海鱼，H murinum，H pubiflorum和黑麦 （图2D）。这种方法的显而易见的好处是良好的传播，无损坏和大量的满足<html> aphase染色体作为成功的FISH分析一个完美的先决条件。能够以存储在细胞悬浮液在-​​20℃至两个月以制备染色体涂在FISH实验当天。新鲜制备的载玻片也可以储存在-20℃下在96％的乙醇，尽管我们观察到杂交信号上这种染色体的质量相比，新制备中期扩展减小。该方法可以用于制备在谷物高质量染色体敷于一种简单，有效的和可再现的方式，最有可能可以在其他植物物种中使用过。 <img alt="图1" src="/files/ftp_upload/53470/53470fig1.jpg" /> 图 1. 计划描述风干滴植物染色体制备方法的过程。 ES / ftp_upload / 53470 / 53470fig2.jpg"/&gt; 图2.鱼大麦 ，H的有丝分裂中期染色体利差球茎和黑麦编写的风干丢弃方法。（A） &#160; H.大麦与单拷贝探针（FPct_40752）标记有红色荧光染料。 （B）的&#160; H.大麦与5S rDNA的探针标记的绿色荧光染料。 （C） &#160; 球茎大麦与CTT-微标记由绿色荧光染料和 （D） &#160; S.黑麦与pSc119.2重复标记的绿色荧光染料。所有染色体进行复用DAPI（红色）。 FISH信号显示为黄色。比例尺= 10微米。 <a href="https://www.jove.com/files/ftp_upload/53470/53470fig2large.jpg" target="_blank">请点击此处查看本figu的放大版本。</a>回覆。 <img alt="表格1" src="/files/ftp_upload/53470/53470tab1.jpg" /> 表1. 孵化酶处理对不同品种的时间。 <img alt="图3" src="/files/ftp_upload/53470/53470fig3.jpg" /> 增刊。图1. 相衬和微分干涉对比（DIC）对 大麦 的例子中，空气干燥滴植物染色体制备方法的有丝分裂中期染色体差图像 。（A）中&#160;相位对比图像 在放大200倍的拍摄 和 （B）在630X的放大倍率采取微分干涉对比图像。 <a href="https://www.jove.com/files/ftp_upload/53470/53470fig3large.jpg" target="_blank">请点击</a>这里查看该图的放大版本。

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

快速风干滴染色体制备方法适用于FISH在植物

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

a-fast-air-dry-dropping-chromosome-preparation-method-suitable-for

Nanyang Technological University

Research

JoVE Journal

Biology

13.2K 次观看.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

视频: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

科研

遗传学

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

斑马鱼胚胎的倍性操作与热休克治疗2

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

发育生物学

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

免疫与感染

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

生物学

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

一种高效的植物多嵌合体荧光融合蛋白的协同表达

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

化学

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和<em在体外施肥

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

快速风干滴染色体制备方法适用于FISH在植物

摘要

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此视频中的章节

使用斑马鱼作为体内模型系统分析 DNA 复制定时

3dpf 斑马鱼幼虫的高通量 DNA 提取及基因分型研究

斑马鱼胚胎的倍性操作与热休克治疗2

产妇基因产品的官能化操作使用

荧

从斑马鱼精子细胞中培养染色体传播

编制果蝇实时成像的胚胎使用悬滴协议

一种高效的植物多嵌合体荧光融合蛋白的协同表达

对于斑马鱼精子超低温冷冻保存的一个高通量的方法和施肥

沉默的火花:CRISPR/Cas9基因组编辑在弱电鱼